家蚕核型多角体病毒几丁质基因的克隆表达及表达产物对Bt杀虫剂的增效作用
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摘要
杆状病毒是鳞翅目昆虫重要的病原微生物，在农业害虫防治中具有很大的应用前景。同时杆状病毒-昆虫(虫体)表达系统是最近发展起来的高效表达外源基因的真核表达系统。因此杆状病毒近年倍受重视，相关的分子生物学研究得到迅速发展。本论文应用现代分子生物学和基因工程技术对家蚕核型多角体病毒(Bombyx mori nucleopolyhedrovirus，BmNPV)几丁质酶基因进行表达和表达产物对Bt杀虫剂的增效作用的研究。
1、从基因组扩增了BmNPV几丁质酶全部编码区基因(chiA)和BmNPV缺失假定信号肽的几丁质酶的编码区基因(chis-)。将chi按其阅读框构建到pET28a原核表达载体T7强启动子下，得到pETchiA重组质粒。再将pETchiA转入大肠杆菌DE3中诱导进行融合表达，通过SDS-PAGE及Western印迹检测，确定表达出分子量为64kD左右的蛋白。薄层扫描显示，表达产物占细胞总蛋白的22％。
2、将PCR扩增出的BmNPV chis-基因构建到杆状病毒-昆虫(Bac-to-Bac)表达系统的供体质粒pFASTBACHTb中，目的基因构建在多角体蛋白基因强启动子控制下，并与6×His tag融合表达。通过转座作用将目的基因重组进穿梭载体Bacmid中，重组Bacmidchis- DNA在Lipofectin介导下共转染粉纹夜蛾(Trichoplusia ni)细胞系(Tn-581-4)。连续传代感染四次后，经PCR检测鉴定转染成功的重组病毒含有目的基因。对感染细胞进行SDS-PAGE分析及Western印迹检测，表明有特异性表达条带，其分子量为63kD左右。薄层扫描显示，表达产物占细胞总蛋白的12％。
3、将以上BmNPV几丁质酶全部编码区基因(chiA)在大肠杆菌和BmNPV缺失假定信号肽的几丁质酶的编码区基因(chis-)在昆虫细胞中的两种表达产物分别加入Bt菌液中一起喂食2龄家蚕，观察并记录不同处理条件下家蚕的死亡时间及死亡时的体重，测算出其LT_(50)和LT_(90)值。实验结果表明，取食了添加BmNPV表达产物的家蚕较对照处理相比，LT_(50)死亡时间明显分别提前24.5 h和25.6 h，体重增量显著偏少。利用TDM模型综合分析，结果表明所得的两种表达产物对Bt杀虫活性具有增效作用。
Baculoviruses are pathogentic to insects of the order Lepidoptera and are attractive biological agents for the control of agriculturally important insect pests. The baculovirus-insects expression system is one of the promising eucaryotic systems in which a lot of foreign genes of economic value have been efficiently expressed. Progress in the field of baculovirus molecular biology have made with more and more concerns taken to study the baculoviruses in the nearing years. This dissertation contains two aspects concerning BmNPV chitinase gene expressed in E. coli and insect cells and the synergistic effect of the expressed products with Bt on killing the insect larvae.
1. The complete chitinase gene (chiA) and the putative signal peptide-deficient encoding region (chis-) were amplified from BmNPVDNA by using PCR method. The target gene (chiA) was inserted into the expression vector pET28a, under the control of phage T7 promoter. Then the recombinant vector pETchiA was transformed into ?coli (DE3) for expression. An expressed band of about 64 kD was identified by SDS-PAGE and further confirmed by Western blot. The expressed target proteins accumulated up to about 22% of the total cellular proteins in the bacterial cells.
2. The putative signal peptide-deficient encoding region of chitinase gene (chis-) was amplified from BmNPV DNA by using PCR method. The target gene (chis-) was inserted into donor plasmid pFASTBACHTb under the control of polyhedin promoter and flanked by the left and right ends of Tn7. The target gene was transposed to the Bacmid in E. coli (DH10), by Tn7 transposition function. Recombinant Bacmid DNA was transfected into Tn-5Bl-4 cell mediated by Lipofectin. An expressed protein band of 63 kD was determined by SDS-PAGE and Western blot. The expressed target protein accumulated up to about 12% of the total cellular proteins in the insect cells.
3. The expression products of those two fragments above from BmNPV mixed with Bt were fed to the second instar larvae of Bombyx mori. After treatment all the lethal time and weights of the killed were recorded, respectively, and the LT50 as well as the LT90 were calculated. The results indicated that lethal time of the larvae of Bombyx mori fed with the Bt added with chitianses from BmNPV expressed in E. coli and Tn cells advanced for 24. 5 and 24. 6 hours, respectively, and the weights augmented were less against the controls. With these results conducted by TMD there was synergistic effect of the expressed products with Bt on killing the insect larvae.
1、从基因组扩增了BmNPV几丁质酶全部编码区基因(chiA)和BmNPV缺失假定信号肽的几丁质酶的编码区基因(chis-)。将chi按其阅读框构建到pET28a原核表达载体T7强启动子下，得到pETchiA重组质粒。再将pETchiA转入大肠杆菌DE3中诱导进行融合表达，通过SDS-PAGE及Western印迹检测，确定表达出分子量为64kD左右的蛋白。薄层扫描显示，表达产物占细胞总蛋白的22％。
2、将PCR扩增出的BmNPV chis-基因构建到杆状病毒-昆虫(Bac-to-Bac)表达系统的供体质粒pFASTBACHTb中，目的基因构建在多角体蛋白基因强启动子控制下，并与6×His tag融合表达。通过转座作用将目的基因重组进穿梭载体Bacmid中，重组Bacmidchis- DNA在Lipofectin介导下共转染粉纹夜蛾(Trichoplusia ni)细胞系(Tn-581-4)。连续传代感染四次后，经PCR检测鉴定转染成功的重组病毒含有目的基因。对感染细胞进行SDS-PAGE分析及Western印迹检测，表明有特异性表达条带，其分子量为63kD左右。薄层扫描显示，表达产物占细胞总蛋白的12％。
3、将以上BmNPV几丁质酶全部编码区基因(chiA)在大肠杆菌和BmNPV缺失假定信号肽的几丁质酶的编码区基因(chis-)在昆虫细胞中的两种表达产物分别加入Bt菌液中一起喂食2龄家蚕，观察并记录不同处理条件下家蚕的死亡时间及死亡时的体重，测算出其LT_(50)和LT_(90)值。实验结果表明，取食了添加BmNPV表达产物的家蚕较对照处理相比，LT_(50)死亡时间明显分别提前24.5 h和25.6 h，体重增量显著偏少。利用TDM模型综合分析，结果表明所得的两种表达产物对Bt杀虫活性具有增效作用。
Baculoviruses are pathogentic to insects of the order Lepidoptera and are attractive biological agents for the control of agriculturally important insect pests. The baculovirus-insects expression system is one of the promising eucaryotic systems in which a lot of foreign genes of economic value have been efficiently expressed. Progress in the field of baculovirus molecular biology have made with more and more concerns taken to study the baculoviruses in the nearing years. This dissertation contains two aspects concerning BmNPV chitinase gene expressed in E. coli and insect cells and the synergistic effect of the expressed products with Bt on killing the insect larvae.
1. The complete chitinase gene (chiA) and the putative signal peptide-deficient encoding region (chis-) were amplified from BmNPVDNA by using PCR method. The target gene (chiA) was inserted into the expression vector pET28a, under the control of phage T7 promoter. Then the recombinant vector pETchiA was transformed into ?coli (DE3) for expression. An expressed band of about 64 kD was identified by SDS-PAGE and further confirmed by Western blot. The expressed target proteins accumulated up to about 22% of the total cellular proteins in the bacterial cells.
2. The putative signal peptide-deficient encoding region of chitinase gene (chis-) was amplified from BmNPV DNA by using PCR method. The target gene (chis-) was inserted into donor plasmid pFASTBACHTb under the control of polyhedin promoter and flanked by the left and right ends of Tn7. The target gene was transposed to the Bacmid in E. coli (DH10), by Tn7 transposition function. Recombinant Bacmid DNA was transfected into Tn-5Bl-4 cell mediated by Lipofectin. An expressed protein band of 63 kD was determined by SDS-PAGE and Western blot. The expressed target protein accumulated up to about 12% of the total cellular proteins in the insect cells.
3. The expression products of those two fragments above from BmNPV mixed with Bt were fed to the second instar larvae of Bombyx mori. After treatment all the lethal time and weights of the killed were recorded, respectively, and the LT50 as well as the LT90 were calculated. The results indicated that lethal time of the larvae of Bombyx mori fed with the Bt added with chitianses from BmNPV expressed in E. coli and Tn cells advanced for 24. 5 and 24. 6 hours, respectively, and the weights augmented were less against the controls. With these results conducted by TMD there was synergistic effect of the expressed products with Bt on killing the insect larvae.
引文
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